|Home | About | Journals | Submit | Contact Us | Français|
Baclofen, a gamma-aminobutyric acid (GABA)B receptor agonist, has been used clinically to treat muscle spasticity, rigidity and pain. More recently, interest in the use of baclofen as an addiction medicine has grown, with promising preclinical cocaine and amphetamine data and demonstrated clinical benefit from alcohol and nicotine studies. Few preclinical investigations, however, have utilized chronic dosing of baclofen, which is important given that tolerance can occur to many of its effects. Thus the question of whether chronic treatment of baclofen maintains the efficacy of acute doses is imperative. The neural substrates that underlie the effects of baclofen, particularly those after chronic treatment, are also not known. In the present study, therefore, rats were treated with either a) vehicle, b) acute baclofen (5 mg/kg) or c) chronic baclofen (5 mg/kg, t.i.d. for 5 days). The effects of acute and chronic baclofen administration, compared to vehicle, were assessed using locomotor activity and changes in brain glucose metabolism (a measure of functional brain activity). Acute baclofen significantly reduced locomotor activity (horizontal and total distance traveled), while chronic baclofen failed to affect locomotor activity. Acute baclofen resulted in significantly lower rates of local cerebral glucose utilization throughout many areas of the brain, including the prefrontal cortex, caudate putamen, septum and hippocampus. The majority of these functional effects, with the exception of the caudate putamen and septum, were absent in animals chronically treated with baclofen. Despite the tolerance to the locomotor and functional effects of baclofen following repeated treatment, these persistent effects on functional activity in the caudate putamen and septum may provide insights into the way in which baclofen alters the reinforcing effects of abused substances such as cocaine, alcohol, and methamphetamine both in humans and animal models.
γ-aminobutyric acid (GABA), the predominant inhibitory neurotransmitter in the brain, achieves its effects through two main receptor subtypes, the ionotropic GABAA and metabotropic GABAB. While the GABAA receptor is a pentameric chloride ion channel, the GABAB receptor is an inhibitory G-protein coupled receptor (Gi/Go) comprised of the GABAB R1A/R1B and GABAB R2 subunits (Möhler et al., 2001). GABAB receptors can serve as pre-synaptic autoreceptors or heteroceptors controlling neurotransmitter release, or as post-synaptic receptors, dampening the excitability of the post-synaptic neuron (Bowery and Enna, 2000). This overall inhibitory effect of GABAB receptor activation results from the opening of inwardly rectifying potassium channels and the inactivation of voltage-gated sodium channels (Bowery and Enna, 2000). GABAB receptors are widely distributed throughout the brain, with dense populations in the amygdala, hippocampus, thalamus and cortex (Bischoff et al., 1999). Impaired GABAergic function has been implicated in a range of conditions such as muscle spasticity, epilepsy, anxiety-disorder and Huntington’s Disease (Wong et al., 2003).
Baclofen, a selective agonist at the GABAB receptor, has been used clinically to treat muscle spasticity and rigidity (Montane et al., 2004) and has shown the potential to treat pain both in animal models (Brusberg et al., 2009; Hama et al., 2012) and clinically (Slonimski et al., 2004). In addition, there has been recent interest recently in the use of baclofen as an addiction medicine. In humans, baclofen has been shown to reduce the number of cigarettes smoked per day (Franklin et al., 2009) and reduce daily alcohol intake in alcohol-dependent individuals (Addolorato et al., 2011). Preclinical data also indicate that baclofen may be useful for treating psychostimulant addiction (Backes and Hemby, 2008; Brebner et al., 2000, 2005; Oleson et al., 2011). Studies have shown that baclofen reduces dopaminergic activity in the nucleus accumbens (Brebner et al., 2005; Fadda et al., 2003; Fu et al., 2012), an important brain region involved in the reinforcing effects of drugs, and it has been shown to reduce drug-motivated behaviors preclinically and clinically. Additionally, acute baclofen dose-dependently reduced responding for d-amphetamine (Brebner et al., 2005) and cocaine (Backes and Hemby, 2008; Brebner et al., 2005) on both fixed- and progressive-ratio schedules.
One of the common limitations in these preclinical studies, however, is the use of a single acute dose of baclofen rather than repeated treatment. This is problematic since baclofen is usually administered chronically in the clinical setting. Thus the question of whether chronic treatment maintains the efficacy of an acute dose in reducing drug-reinforced behavior is important. Indeed, it has been well recognized that tolerance to the effects of baclofen occurs. Multiple clinical and preclinical studies have reported reductions in the effects of baclofen following chronic treatment and the requirement of increasing doses to maintain its therapeutic effect, i.e. the development of tolerance (Dones and Broggi, 2010; Heetla et al., 2009; Lehman et al., 2003; Soni et al., 2003). A separate question is what are the underlying neural substrates of the effects of baclofen? Can we identify the brain regions involved in the functional effects of baclofen, and does activity change following repeated treatment? The answers to these questions are important since they will help us better understand how baclofen acts in the brain and how useful it and similar compounds may be as substance abuse medications. Additionally, given the widespread distribution of GABAB receptors and the variety of behavioral effects of baclofen, whole brain neuroimaging methods may be particularly well-suited for identifying the neural substrates of its actions. The present study was designed, therefore, to determine the influence of the acute administration of baclofen on functional activity within the brain, using the 2-[14C]-deoxyglucose method, and assess whether tolerance to these effects occurred with repeated baclofen treatment. In order to have an additional measure by which to determine the degree of any behavioral tolerance that developed, we also measured locomotor activity in the same animals. Acute treatment with GABAB agonists is well known to produce decreases in locomotor activity (Frankowska et al., 1999; Liang et al., 2006; Paredes and Almo, 1989) and thus behavioral measures can provide an index of the development of tolerance.
All animal procedures were performed in accordance with protocols approved by Wake Forest University School of Medicine Animal Care and Use Committee and were consistent with the NIH Guide for the Care and Use of Laboratory Animals. Male Sprague-Dawley rats (280-300 g) were obtained from Harlan Industries (Indianapolis, IN). All animals were maintained in a temperature-and humidity-controlled vivarium with a12-h light-dark cycle (lights on at 07:00). Food and water were available ad libitum.
Surgical procedures for the 2-[14C]-DG studies followed those described by Torres-Reveron et al. (2006) and were carried out 24 hours before the 2DG procedure to allow for anesthetic clearance and recovery. Briefly, rats were lightly anesthetized with a mixture of isoflurane and nitrous oxide and catheters were implanted in the jugular vein. Catheters were filled with heparinized saline and run subcutaneously to exit at the nape of the neck. Surgery lasted no longer than 45 min, following which animals were returned to their home cages for recovery. Animals were food-deprived for 8 hours (overnight) before the initiation of the 2DG procedure.
Baclofen (Sigma Aldrich, St. Louis, MO) was dissolved in saline and injected via the intraperotineal (i.p.) route in a 1 ml/kg volume. Animals were administered either baclofen or vehicle 3 times daily (ter in die (t.i.d.), 5 mg/kg) for 5 consecutive days, followed by an additional injection on the morning of day 6. The dose of baclofen was chosen based on data from previous studies demonstrating the ability of this dose to block the development of cocaine sensitization (Frankowska et al., 2009) and reduce responding for amphetamine on a fixed-ratio and progressive ratio schedule (Brebner et al., 2005). We chose to administer baclofen t.i.d. due to its relatively short half-life, which has been estimated to range from 4.58 (Anderson et al., 1984) to 6.8 hours (Wuis et al., 1989).
Prior to any drug treatment, all animals (n = 24) were habituated to locomotor chambers for two daily 2 h sessions (Day 1 and 2; Table 1) prior to baseline locomotor testing (Day 3). Locomotor activity was measured in open-field Plexiglas® test chambers (42 × 42 × 30 cm) by electronic counters that detected interruptions of 8 independent infrared photocell beams (Omnitech, Columbus, OH). Photocell counts were recorded for 15 min and the following measures were calculated: horizontal activity and total distance travelled. After measurement of baseline locomotor activity, animals were assigned to one of 3 groups, matched for levels of baseline locomotor activity, 1) control (n = 8), 2) acute baclofen (n = 8) and 3) chronic baclofen (n = 8). On the final test day (Day 9), animals were administered either saline or baclofen (5 mg/kg, i.p.) 15 min prior to being placed in the locomotor chamber. Locomotor activity was recorded for 15 min, then the 2-[14C]-deoxyglucose method was initiated (see below). Given the relatively short half-life and duration of action of baclofen, locomotor activity was recorded for 15 min in order to capture the maximum effect without interference of blood sampling necessary for the 2DG procedure. Previous data have shown baclofen to be behaviorally active after a similar pretreatment time (Paredes and Agmo, 1989). This allowed both procedures to occur within the timeframe of maximal drug effect without interference with the measurement of either.
Local cerebral glucose utilization was measured according to the method of Sokoloff et al. (1977), as adapted for use in freely moving animals (Crane and Porrino, 1989; Torres-Reveron et al. 2006). Jugular catheters were utilized in order to prevent any reduced mobility that might result from surgery of the femoral artery and vein thus interfering with the measurement of spontaneous locomotor activity. This modification of the 2DG procedure using the jugular to collect timed blood samples has been verified to provide similar results as those obtained with arterial sampling (Torres-Reveron et al. 2006). Surgeries took place on the morning of Day 8 (5th day of drug treatment) and took no longer than 45 min to complete (including induction and recovery from anesthesia), ensuring that the next two injections of baclofen or vehicle could take place that day. 24 h after the surgery, on the final test day (Day 9 or immediately after the final drug dose), rats were given an intravenous pulse of 2-[14C]deoxyglucose (75 μCi/kg; specific activity 50–55 mCi/mmol; Perkin Elmer, Waltham, MA) and the catheter was flushed with 0.2 ml saline. Timed venous blood samples were then drawn over the next 45 min by applying slight negative pressure on a 1 ml syringe. The catheter was flushed with saline after each blood sample was taken. Samples were centrifuged and plasma concentrations were measured for 2[14C]DG and glucose. At the end of the 45-min sampling procedure, animals were sacrificed using sodium pentobarbital (100 mg/kg, i.v.). Brains were rapidly removed and frozen in isopentane at −45°C, then stored at −80°C until sectioning.
Coronal sections (20 μm) were cut in a cryostat maintained at −22°C. Sections were picked up on glass coverslips, dried on a hotplate (60°C), and then exposed to film (Kodak MRM film, Kodak, Rochester, New York) along with calibrated [14C] standards (Amersham, Arlington Heights, IL) for 12–15 days. Autoradiograms were then analyzed by quantitative densitometry with a computerized image processing system (MCID Imaging Research, Cambridge, England). Optical density measurements for each brain structure were made in a minimum of five sections. Local cerebral glucose utilization was calculated using the operational equation defined by Sokoloff et al. (1977). Rates of glucose utilization were determined in 35 brain structures selected on the basis of previous reports (Whitlow et al., 2002); according to the rat brain atlas (Paxinos and Watson, 1997).
Measures of locomotor activity were analyzed by means of one way analyses of variance (ANOVA) followed by planned Bonferroni’s test for multiple comparisons. Global rates of glucose utilization were calculated as the weighted average of rates of glucose metabolism across analyzed brain regions. Global rates of glucose utilization were then compared using one way ANOVA. Rates of local cerebral glucose utilization were analyzed in specific functional groups of brain regions (cortex, basal ganglia, limbic, thalamus, midbrain) using two way ANOVA, brain region group × treatment (vehicle vs. acute baclofen vs. chronic baclofen) with brain region group as a repeated measure. These were followed by planned Bonferroni’s tests for multiple comparisons. Statistical significance was considered as p < 0.05.
The effects of acute and chronic baclofen treatment on locomotor activity are shown in Figure 1. When compared to vehicle treatment, the acute administration of baclofen (5 mg/kg) significantly reduced spontaneous locomotor activity. Measures of both total distance traveled and horizontal movement of the baclofen treated animals were significantly lower than those of vehicle treated rats. In contrast, chronic baclofen administration (5 mg/kg, t.i.d. for 5 days) did not significantly alter locomotor activity as compared to controls (see Figure 1).
Local rates of cerebral glucose utilization of vehicle-treated, acute baclofen- and chronic baclofen-treated animals are shown in Table 2 and Figure 2. Global rates of glucose utilization were significantly lower in animals treated acutely with baclofen, compared to vehicle treated controls (mean ± S.E.M., 86.6 ± 3.8 μmol/100 g/min, controls vs. 68.3 ± 2.9 μmol/100 g/min, acute baclofen, p <0.001). In contrast, global rates of glucose utilization in animals chronically treated with baclofen were not significantly different from controls (86.6 ± 3.8 μmol/100 g/min, controls vs. 82.7 ± 3.5 μmol/100 g/min, chronic baclofen).
Two way ANOVAs were used to compare rates of glucose utilization (drug condition × brain region group). In the cortex, there were significant main effects of drug condition (F(2, 13) = 10.62, p = 0.002) and brain region (F(6, 78) = 63.29, p < 0.001), as well as a significant interaction (F(12, 78) = 3.33, p = 0.001). Multiple comparisons showed that acute baclofen administration resulted in significantly lower rates of glucose utilization throughout many portions of the cortex, compared to vehicle treated controls. These regions included prelimbic (− 23%), infralimbic (− 20%), agranula insula (− 15%), anterior cingulate (− 20%), posterior cingulate (− 26%) and motor (− 13%) cortex.
Within the basal ganglia, there were main effects of drug condition (F(2, 12) = 20.93, p < 0.001) and brain region (F(7, 84) = 160.62, p < 0.001), as well as a significant interaction (F(14, 84) = 3.38, p < 0.001). Multiple comparisons showed that acute baclofen treatment resulted in significantly lower functional activity in the dorsomedial caudate (− 26%), dorsolateral caudate (− 22%), putamen (− 21%), rostral nucleus accumbens (− 21%), nucleus accumbens core (− 25%), substantia nigra pars compacta (− 35%), substantia nigra pars reticulata (− 29%) and subthalamic nucleus (− 30%), compared to controls.
Within limbic regions, there were significant main effects of drug condition (F(2, 13) = 3.94, p < 0.05) and brain region (F(10, 130) = 83.94, p < 0.001), as well as a significant interaction (F(20, 130) = 4.21, p < 0.001). Multiple comparisons showed that rates of glucose utilization were significantly lower following acute baclofen treatment, compared to controls, in the lateral septum (−21%), medial septum (−36%) and hippocampal CA3 region (−23%). Within the thalamus, there were significant main effects of drug condition (F(2, 13) = 7.78, p < 0.01) and brain region (F(3, 39) = 8.91, p < 0.001), but no significant interaction. Multiple comparisons showed that significantly lower levels of functional activity were present in the paraventricular (−26%), lateral (−20%) and ventral (−21%) nuclei. Within the midbrain and brainstem, there were significant main effects of drug condition (F(2, 13) = 11.20, p < 0.001) and brain region (F(18, 234) = 50.673, p < 0.001), but no significant interaction. Multiple comparisons showed that rates of glucose utilization in the lateral geniculate (−23%), superior colliculus (−25%), dorsal periaqueductal grey (−31%) and medial raphe (−32%) were significantly lower after acute baclofen administration, compared to controls.
In contrast, multiple comparisons revealed that rates of glucose utilization of rats receiving chronic administration of baclofen did not significantly differ from those of vehicle-treated rats in the majority of brain regions examined. Chronic baclofen treatment did, however, significantly reduce rates of glucose utilization in dorsomedial caudate (−8%), dorsolateral caudate (−14%) and lateral septum (−20%), as compared to vehicle-treated controls.
The data from the present study demonstrate that acute administration of baclofen reduced rates of glucose metabolism throughout a diffuse network of brain areas, compared to controls, consistent with the decreased levels of spontaneous locomotor activity seen in the same animals. In contrast to these widespread reductions in metabolic activity, chronic baclofen administration resulted in lower rates of glucose utilization in a very restricted pattern of brain areas, as compared to those of saline-treated controls. This is consistent with the tolerance that developed to the effects of baclofen on locomotor activity seen here, as well as reports of tolerance to the other behavioral effects that accompany baclofen treatment. There was, however, a subset of structures, mainly within the nigrostriatal and mesolimbic systems, in which baclofen significantly altered functional activity even after repeated baclofen treatment.
The finding that acute baclofen treatment reduced locomotor activity agrees with a substantial body of literature demonstrating a motor impairing effect of baclofen (Lobina et al., 2005; Paredes and Agmo, 1989; Van Nieuwenhuijzen et al., 2009) and is in line with its clinical usage as a muscle relaxant (Richard and Menei, 2007). For example, 5 mg/kg baclofen (the dose used in the current study) has been shown to acutely inhibit locomotor activity by approximately 30 – 60% (Frankowska et al., 2009; Paredes and Agmo, 1989). GABA has been shown to directly inhibit dopamine-containing neurons in the substantia nigra via the striatonigral and pallidonigral pathways (Bunney and Aghajannian, 1976; Kubota et al., 1987). Moreover, since GABAB receptors have been shown to be located in the substantia nigra (Bowery et al., 1987; Boyes and Bolam, 2003), it is possible that elevated levels of GABAergic activity resulted in less dopaminergic activity via inhibition of nigral dopamine cells, thereby suppressing levels of locomotor activity (Beninger, 1983).
Our findings of lower levels of functional brain activity following acute baclofen treatment are also in line with previous studies utilizing a different GABAB agonist, γ-hydroxybutyric acid (GHB; Crosby et al., 1983; Kuschinsky et al., 1985). Kuschinsky and colleagues reported lower rates of glucose utilization following acute GHB administration (Kuschinsky et al., 1985), in a similarly distributed network to that reported here following acute baclofen. For example, functional activity in the frontal cortex, caudate, substantia nigra, thalamus and hippocampus was lower in GHB-treated animals, compared to controls. Though GHB is generally regarded as a GABAB agonist, it has been suggested that GHB may also interact with its own distinct receptor (Andriamampandry et al., 2003, 2007). Regardless, the overlap of brain areas affected by GHB and baclofen, and the selectivity of baclofen for the GABAB receptor would suggest that many of the effects of GHB may be mediated by GABAB receptors.
The brain regions in which significant changes in functional activity following acute baclofen were measured also correlate well with the known distribution of the GABAB receptor (Bischoff et al., 1999; Bowery et al., 1983). There are three splice variants of the GABAB receptor – the R1A and R1B variants (Kaupmann et al., 1997, 1998a) and R2 variant (Kaupmann et al., 1998b). GABAB receptors are heteromers formed from the GABAB R1A or R1B and R2 variants in a stochiometry of 1:1 (Kaupmann et al., 1998). Bischoff et al. (1999) investigated the relative distribution and density of the GABAB R1A and R1B variants throughout the rat brain. While the highest densities of the R1A variant were located in the substantia nigra, ventral tegmental area, periaqueductal grey, hippocampus and cortex, the R1B variant was more restricted in its distribution with higher concentrations in the thalamus, habenula and cerebellum (Bischoff et al., 1999). As expected, since the GABAB R2 variant is required for a functional GABAB receptor, the distribution of the R2 variant largely parallels that of the R1A and R1B (Möhler et al., 2001). Interestingly, many of the brain areas in the current study that exhibited the greatest degree of tolerance to the functional effects of baclofen (for example substantia nigra, hippocampus, thalamus and cerebellum) also contain the highest densities of GABAB receptors (Bischoff et al., 1999), while regions with more moderate levels of GABAB receptors, such as the caudate putamen, exhibited a lesser degree of tolerance. Finally, it is tempting to speculate on some of the functional outcomes of baclofen-induced decreases in rates of glucose metabolism in specific brain areas. For example, perhaps the ability of baclofen to relax muscles stems from its effects within brain regions such as the caudate putamen, globus pallidus and cerebellum, while its pain relieving effects may result from activity in regions such as the dorsal periaqueductal grey.
In contrast to the acute effects of baclofen on locomotor activity and functional activity, chronic treatment with baclofen failed to significantly alter either ambulatory behavior or rates of glucose utilization in the majority of brain areas measured. These data suggest that tolerance developed over the 5 days of repeated injections. Previously, evidence of the development of tolerance to the effects of baclofen has come from both clinical and preclinical reports (Akman et al., 1993; Gianutsos and Moore, 1978; Hefferan et al., 2006; Soni et al., 2003) and we have previously shown the 2DG method to be sensitive to drug-induced tolerance to the metabolic effects of drugs (Whitlow et al., 2003). However, studies have shown that tolerance does not develop to all the central and physiological effects of baclofen. For example, the antinociceptive effect of acute baclofen remains following chronic treatment, but not the acute locomotor effects (Levy and Proudfit, 1977).
Though the underlying molecular mechanism involved in the development and expression of this tolerance is not certain, it is possible that repeated baclofen treatment may alter the density, or result in desensitization, of GABAB receptors (Kohout and Lefkowitz, 2003). Indeed, previous studies have shown that the density and functional response of GABAB receptors can decrease following prolonged treatment with selective agonists, such as baclofen (Malcangio et al., 1993) or, conversely, increase following chronic treatment with specific antagonists (Malcangio et al., 1993; Pirbri et al., 2005). However, there is evidence both for, and against, the involvement of GABAB receptor alterations in the development of tolerance to baclofen. For example, Sands et al. (2003) reported tolerance to the sedative effects of baclofen over 7 days of treatment, which was accompanied by GABAB receptor desensitization, as measured by [35S] GTPyS binding. In contrast, Lehmann et al. (2003) demonstrated that tolerance developed to the hypothermic effects of baclofen following repeated treatment, but this was not associated with significant changes in either GABAB receptor density or mRNA expression. Either way, it is apparent from Fairfax et al. (2004) that the mechanism of baclofen-induced GABAB receptor degradation is quite different from other classic G-protein coupled receptors and tolerance may occur to some effects of baclofen and not to others.
Although tolerance developed to the depressant effects of baclofen administration in a wide range of brain regions, persistent significant decreases in functional activity were observed in the striatum and septum, brain areas that have been shown to play an important role in the regulation of reward, reinforcement and habit formation (Balleine, 2005; Dalley and Everitt, 2009; Taber et al., 2012). In addition, there were trends towards continued decreases in the medial forebrain bundle and ventral tegmental area. That functional activity remains depressed in these portions of the mesolimbic system suggests that the effects of baclofen in these areas persist even after prolonged administration; and may, therefore, be the substrates of its ability to reduce the reinforcing effects of a variety of abused drugs, such as alcohol, cocaine, amphetamine and nicotine (Addolorato et al., 2011; Brebner et al., 2000, 2005; Fattore et al., 2009;). These effects are also consistent with those reported in a recent study by Franklin et al. (2011). These authors measured resting brain cerebral blood flow in humans before and after 21 days of baclofen treatment and found that baclofen resulted in significant decreases in blood flow in the ventral striatum, medial orbitofrontal cortex and insula. The persistent functional effects of baclofen in these limbic areas, even after chronic dosing, suggests that baclofen may have potential as an effective addiction medicine, and that it may remain efficacious even with long term treatment.
In summary, the present report describes the effects of the administration of baclofen on rates of local cerebral glucose utilization. Acute baclofen administration at a dose that depressed spontaneous locomotor activity produced widespread decreases in functional activity throughout the central nervous system. In contrast, after chronic treatment, baclofen administration no longer depressed locomotor activity and, in the majority of brain regions, rates of glucose utilization were no longer different from control rates. However, rates of glucose utilization in the striatum and septum remained decreased at levels similar to those following acute baclofen administration. These persistent effects on functional activity may provide insights into the way in which baclofen alters the reinforcing effects of abused substances such as cocaine, alcohol, and methamphetamine both in humans and animal models.
The authors wish to thank Mack Miller for his technical help. This research was funded by NIDA grant P50 DA06634.
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.